AB3563 Sigma-AldrichAnti-phospho mouse HSP25 (pS86) Antibody

Homeostasis of circulating cortisol is maintained by the 11β-HSD2 enzyme which inactivates cortisol into cortisone. It is abundantly expressed in the placenta where it protects the fetus from high levels of maternal glucocorticoids (GCs). Maternal administration of Carbenoxolone (Cbx), a powerful 11β-HSD2 inhibitor, leads to an increase in fetal cortisol. Previous data showed that intrauterine environment plays a crucial role in determining hippocampal structure and function. Exposure of pregnant rats to high levels of GC leads to low birth weight in offspring and an increased risk of age related memory and cognitive deficits later in life. Glutamate receptors are localized in the postsynaptic density (PSD), where many signaling proteins, cytoskeleton proteins, and ion channels are found. Any change in the number of these molecules can influence the morphology and function of the dendritic spine. We proposed that repeated Cbx injections during late pregnancy may alter the scaffolding proteins of the NMDA receptor in the pup\'s brain. We investigated the effects of repeated maternal Cbx injections on the scaffolding proteins of NMDA receptor in the hippocampus of rat pups. We showed that injecting pregnant rats with Cbx injections (30mg/kg) during GD 14-21 leads to a significant decrease in SPAR (Spine Associated Rap Guanylate kinase activating protein) (p<0.001) and PSD-95 (p<0.05) but a significant increase in Snk (Serum inducible kinase) (p<0.001) in the pup\'s hippocampus at P40. In general, Snk is induced by neuronal activity and plays an important role in phosphorylating SPAR. The phosphorylated SPAR is then recognized and degraded by ubiquitin proteasome system (UPS), causing the depletion of SPAR and PSD-95 from the spines. The results suggest that fetal exposure to excessive GC levels may activate the Snk/SPAR pathway and lead to the depletion of SPAR and PSD-95. Since GCs drugs are commonly used in various obstetric and pediatric conditions, it is important to consider the risks and benefits of prenatal GCs exposure in order to prevent neurodevelopmental delay in the offspring.Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Renal replacement therapy by peritoneal dialysis is frequently complicated by technical failure. Peritoneal dialysis fluids (PDF) cause injury to the peritoneal mesothelial cell layer due to their cytotoxicity. As only isolated elements of the involved cellular processes have been studied before, we aimed at a global assessment of the mesothelial stress response to PDF. Following single or repeated exposure to PDF or control medium, proteomics and bioinformatics techniques were combined to study effects in mesothelial cells (MeT-5A). Protein expression was assessed by two-dimensional gel electrophoresis, and significantly altered spots were identified by MALDI-TOF MS and MS2 techniques. The lists of experimentally derived candidate proteins were expanded by a next neighbor approach and analyzed for significantly enriched biological processes. To address the problem of an unknown portion of false positive spots in 2DGE, only proteins showing significant p-values on both levels were further interpreted. Single PDF exposure resulted in reduction of biological processes in favor of reparative responses, including protein metabolism, modification and folding, with chaperones as a major subgroup. The observed biological processes triggered by this acute PDF exposure mainly contained functionally interwoven multitasking proteins contributing as well to cytoskeletal reorganization and defense mechanisms. Repeated PDF exposure resulted in attenuated protein regulation, reflecting inhibition of stress responses by high levels of preinduced chaperones. The identified proteins were less attributable to acute cellular injury but rather to specialized functions with a reduced number of involved multitasking proteins. This finding agrees well with the concept of conditioning effects and cytoprotection. In conclusion, this study describes the reprogrammed proteome of mesothelial cells during recovery from PDF exposure and adaption to repetitive stress. A broad stress response with a number of highly overlapping processes and multitasking proteins shifts toward a more specific response of only few less overlapping processes.

Thyroxine pretreatment increases the tolerance of the heart to ischaemia, and heat-shock protein 27 (HSP27) is considered to play an important role in cardioprotection. The present study investigated whether long-term thyroxine administration can induce changes in the expression, translocation and phosphorylation of HSP27 at baseline and upon ischaemic stress. L-Thyroxine (T(4)) was administered to Wistar rats (25 microg/100 g/day s.c.) for 2 weeks, while normal animals served as controls. Hearts from normal and thyroxine-treated rats were perfused in Langendorff mode and subjected to 10 or 20 min of zero-flow global ischaemia only or to 20 min of ischaemia followed by 45 min of reperfusion. Total and phospho-HSP27 expression were assessed at different times in the Triton-soluble (cytosol-membrane), S fraction, and the Triton-insoluble (cytoskeleton-nucleus) fraction, P fraction. Postischaemic recovery of left ventricular developed pressure at 45 min of reperfusion was expressed as % of the initial value. In hearts from thyroxine-treated animals, the levels of basal total HSP27 and phospho-HSP27 in the P fraction were significantly increased as compared to normal. In response to ischaemia, in hearts from thyroxine-treated rats, the levels of total HSP27 and phospho-HSP27 were found to be significantly increased in the P fraction at 10 and 20 min of ischaemia as compared to preischaemic values, whereas in normal hearts, the levels of total HSP27 and phospho-HSP27 were significantly increased at 20 min only. Postischaemic functional recovery was significantly greater in thyroxine-treated than in untreated hearts. In summary, long-term thyroxine pretreatment results in an increased basal expression and phosphorylation of HSP27 and in an earlier and sustained redistribution of HSP27 from the S to the P fraction in response to ischaemia. This effect might be of important therapeutic relevance.

Activation of the serine-threonine kinase Akt by cytokines, chemokines, and bacterial products delays constitutive neutrophil apoptosis, resulting in a prolonged inflammatory response. We showed previously that Akt exists in a signaling complex with p38 MAPK, MAPK-activated protein kinase-2 (MAPKAPK-2), and heat shock protein-27 (Hsp27); and Hsp27 dissociates from the complex upon neutrophil activation. To better understand the regulation of this signaling module, the hypothesis that Akt phosphorylation of Hsp27 regulates its interaction with Akt was tested. The present study shows that Akt phosphorylated Hsp27 on Ser-82 in vitro and in intact cells, and phosphorylation of Hsp27 resulted in its dissociation from Akt. Additionally, the interaction between Hsp27 and Akt was necessary for activation of Akt in intact neutrophils. Constitutive neutrophil apoptosis was accelerated by sequestration of Hsp27 from Akt, and this enhanced rate of apoptosis was reversed by introduction of constitutively active recombinant Akt. Our results define a new mechanism by which Hsp27 regulates apoptosis, through control of Akt activity.